A high-throughput screen is proposed for the identification of inhibitors of the influenza NS1 protein. Influenza is a world-wide public health problem and emerging forms of the virus have the potential to cause a pandemic of equal or greater magnitude to the outbreaks recorded in 1918, 1957 or 1968. Vaccine development is proceeding and there also exist two classes of anti-influenza compounds. However these therapeutic modalities are neither fully effective nor widely enough available to fulfill global needs. In addition their potential usefulness against newly emergent strains is not known. Efforts are needed to develop novel agents against influenza virus, including broad-spectrum agents. Identification of small molecules that inhibit NS1 function either directly or by interfering with specific cellular pathways may be a key to increasing our defense against the virus. A yeast-based assay for NS1 function will be used to screen for novel inhibitors. NS1 induces a pronounced slow-growth phenotype that can be specifically suppressed by small molecule inhibitors. The growth assay has been converted to a 96 well format and it has been shown to be of high quality. A small scale screen of 2000 compounds from the NCI Diversity Set identified several inhibitors of NS1 function that also inhibit influenza virus replication in cell culture. The inhibitors block the ability of NS1 to prevent activation of the interferon response. Some of them also reverse the ability of NS1 to interfere with nuclear-to- cytoplasmic transport of cellular RNA. The high-throughput screen proposed here is expected to identify compounds of higher potency and greater structural diversity than those identified from the small scale screen. This will shed light on the mechanisms of NS1 function and provide starting points for the development of clinically useful agents. PUBLIC HEALTH RELEVANCE: Influenza is a world-wide public health problem of major proportions. The likelihood of a devastating influenza pandemic during this century makes it essential to discover new drugs to combat the disease. The proposed high throughput screen for small molecule inhibitors of the NS1 protein from influenza virus will explore the possibility that this protein can be used as a target for clinically useful, broad spectrum therapeutics against influenza virus. [unreadable] [unreadable] [unreadable]